CN114778734B - Quantitative determination method and application of polar lipid - Google Patents

Quantitative determination method and application of polar lipid Download PDF

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CN114778734B
CN114778734B CN202210429536.XA CN202210429536A CN114778734B CN 114778734 B CN114778734 B CN 114778734B CN 202210429536 A CN202210429536 A CN 202210429536A CN 114778734 B CN114778734 B CN 114778734B
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volume ratio
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王兴国
江晨瑜
韦伟
金青哲
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Jiangnan University
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Abstract

The invention discloses a quantitative determination method of polar lipid and application thereof, and belongs to the technical field of detection and analysis. The method specifically comprises the following steps: extracting total lipid containing polar lipid, enriching and purifying polar lipid, and performing supercritical fluid chromatography-mass spectrometry detection analysis on the polar lipid enriched substance after redissolution. The detection method can realize unified determination of cholesterol, phospholipid and ganglioside, optimize solid phase extraction conditions so that cholesterol and sphingomyelin can be reserved in polar lipid eluent, and realize rapid qualitative and quantitative detection. The result of the measurement by the internal standard method is accurate, and the relative values of the peak areas of the internal standard substance and the measured component are measured to calculate, so that errors caused by changes of operating conditions, loading volumes and the like are eliminated to a certain extent.

Description

Quantitative determination method and application of polar lipid
Technical Field
The invention belongs to the technical field of detection and analysis, relates to a detection method of polar lipids, and particularly relates to a method for detecting polar lipids by using a supercritical fluid chromatography-tandem mass spectrometry.
Background
Polar lipids include phospholipids, cholesterol, ceramides, gangliosides, and the like. Although the content of polar lipids is low in some biological samples (e.g. only 2% of milk fat), it is important in terms of biological and physiological functions. Polar lipids determine the integrity of cell membranes and are involved in cell signaling, proliferation, inflammatory responses, and the like. In breast milk, polar lipids are mostly distributed on Milk Fat Globule Membrane (MFGM). Cholesterol is the major cholesterol lipid of breast milk and is also an important component of MFGM. Recent studies have shown that the composition of the polar lipids of breast milk is very complex, and that some of the polar lipids exist in milk in fat globules with milk fat highly correlated to cognitive development of infants, with the core triglycerides surrounded by membranes composed externally of polar lipids such as phospholipids, cholesterol, etc., thus keeping the whole emulsion system stable. Cholesterol is an important component of cell membranes, phospholipids in milk are associated with infant brain and intestinal immune system development, gastrointestinal tract infection protection and memory improvement in the elderly, phosphatidylcholine (PC) and Sphingomyelin (SM) are important polar lipids, and are structural components required for rapid growth of organs and cell membranes.
Due to the very low polar lipid content in some biological samples. Therefore, in some detection processes, other lipid substances such as high-content triglyceride cause interference to the detection of polar lipids, which greatly increases the difficulty in analyzing polar lipids with complex composition and large property differences. Thus, enrichment and purification of polar lipids is often required.
Solid Phase Extraction (SPE) is one of the effective methods of sample preparation, the basic principle of which is similar to liquid-liquid extraction, which is capable of adsorbing and enriching and purifying analytes on solid adsorbent plugs from a sample solution. For enrichment and purification of polar lipids, currently existing patents and literature are directed to enrichment of phospholipids, however, most of the existing methods have the problem of loss of sphingomyelin and cholesterol. Cholesterol is more polar than phospholipids than triglycerides, making separation and purification difficult. In addition, although the prior patent CN106153763a separates and purifies the lipid with relative polarity by liquid, the method has the problems of complex operation, easy loss of cholesterol and sphingomyelin, large milk fat consumption of single sample, and the like.
In the related art, there are few reports that the composition of cholesterol, phospholipid and ganglioside can be quantitatively detected at the same time, which often requires a plurality of different detection methods, and thus data cannot be unified and the consumption of samples is increased, so that the detection of some rare samples is limited. In addition, the chromatographic detection method of the polar lipid in the related art generally has long time, so that the accurate qualitative determination of the molecular species of the polar lipid is difficult to realize.
Disclosure of Invention
Accordingly, in order to overcome the above-mentioned drawbacks of the related art, a first object of the present invention is to provide a method for detecting polar lipids, comprising the steps of:
(1) Extracting total lipid containing polar lipids:
Adding an organic solvent A into emulsion raw materials, dairy products or plasma, shaking and mixing uniformly, adding an internal standard solution B, adding an organic solvent C, shaking and mixing uniformly, then carrying out ultrasonic treatment, carrying out ice water bath ultrasonic treatment for 5-20 minutes, adding a liquid D, shaking and mixing uniformly, and promoting layering to obtain a mixture I;
Centrifuging the obtained mixture I, removing the upper organic solution, and removing water and solvent by using a vacuum centrifugal concentrator to obtain a total lipid extract containing polar lipids;
Wherein the organic solvent A is methanol; the internal standard solution B is an isotope internal standard mixed solution, and the organic solvent C is methyl tertiary butyl ether; the liquid D is ultrapure water;
(2) Enriching and purifying polar lipids:
Firstly, carrying out activation treatment on a solid phase extraction column, wherein the mass of silica gel filled in the solid phase extraction column is 200-1000 mg, the volume of the column is 3-6 mL, and n-hexane is used for activation;
Dissolving 5-200 mg of the total lipid extract containing polar lipids in an organic solvent E to obtain a sample solution, and loading the sample solution onto an activated solid phase extraction column;
Eluting the column with an organic solvent F and an organic solvent G in sequence to obtain neutral fat eluent containing triglyceride;
eluting the column with organic solvent H, organic solvent I and organic solvent J sequentially to obtain polar lipid-rich eluate, and drying with nitrogen to obtain polar lipid concentrate
Wherein, the organic solvent E is chloroform: methanol is mixed according to the volume ratio of 1:1-8:1; the organic solvent F is n-hexane: the diethyl ether is mixed solution with the volume ratio of 100:1-5:1; the organic solvent G is n-hexane: the diethyl ether is mixed solution with the volume ratio of 10:1-2:1; the organic solvent H is n-hexane: the diethyl ether is mixed solution with the volume ratio of 3:1-0.5:1; the organic solvent I is methanol; the organic solvent J is chloroform: methanol: mixing liquid with water according to the volume ratio of 3:5:2;
(3) After redissolving the polar lipid enriched material, carrying out supercritical fluid chromatography-mass spectrometry detection analysis:
The redissolution solvent used for redissolution of the polar lipid enrichment is chloroform: the mass volume ratio of the polar lipid enrichment to the redissolution solvent is 1.5mg to 1mL to 10mg to 1mL according to the volume ratio of the mixed solution of methanol 2:1.
The detection method can realize unified determination of cholesterol, phospholipid and ganglioside, optimize solid phase extraction conditions so that cholesterol and sphingomyelin can be reserved in polar lipid eluent, and realize rapid qualitative and quantitative detection.
The result of the measurement by the internal standard method is accurate, and the relative values of the peak areas of the internal standard substance and the measured component are measured to calculate, so that errors caused by changes of operating conditions, loading volumes and the like are eliminated to a certain extent.
In the step (1), the target substance is distributed on the upper layer by the collocation of methanol and methyl tertiary butyl ether, so that the extraction operation is convenient, and the method is particularly suitable for experiments of large samples.
The second object of the present invention is to provide the use of a method for quantitative determination of polar lipids in emulsion raw materials, dairy products or plasma samples.
Optionally, the conditions of the supercritical fluid chromatography-mass spectrometry detection analysis are as follows:
Supercritical fluid chromatography with water-based ultra-high performance liquid chromatography UPC 2, water-based UPC 2 BEH,150mm×3.0mm,1.7 μm, water, USA; the column temperature is 55 ℃, the back pressure is 1500psi, and the sample injection volume is 2 mu L; the eluent A is supercritical CO 2, the purity is more than or equal to 99.99%, the eluent B is methanol/water solution containing ammonium formate, the volume percentage of the methanol is 90% -100%, the content of the ammonium formate is 5 mM-35 mM, the elution gradient is 0min, and the content of the ammonium formate is 5% B;5.5min,48% B;10min,48% b;
the mass spectrum adopts WATERS SYNAPT Q-TOF-MS mass spectrometer, the flow rate of collision gas (argon) is 50L/h, the flow rate of desolvation gas (nitrogen) is 700L/h, the ion source temperature is 100 ℃, the desolvation temperature is 400 ℃, the cone hole voltage is 30eV, the low-energy collision is set to be 6eV, the high-energy collision is set to be 20-45eV, and the mass-to-charge ratio scanning range is 100-1600m/z.
Optionally, the isotopic internal standard mixed solution contains: 30 μg/mL Cer (d18:1-D7/15:0), 5 μg/mL CL (18:2/18:2/18:2/18:2) -D5, 100 μg/mL cholesterol -D7,30μg/mL PG(15:0/18:1-D7),5μg/mL PE(15:0/18:1-D7),10μg/mL PI(15:0/18:1-D7),5μg/mL LPE 18:1-D7,7μg/mL PA(15:0/18:1-D7),5μg/mL PS(15:0/18:1-D7),160μg/mL PC(15:0/18:1-D7),30μg/mL SM(d18:1-D9) and 25 μg/mL LPC 18:1-D7.
Optionally, the volume ratio of the emulsion raw material, the dairy product or the plasma to the methanol is 1:2-1:8; the volume ratio of the emulsion raw material, the dairy product or the blood plasma to the internal standard solution B is 20:1-80:1; the volume ratio of the emulsion raw material, the dairy product or the blood plasma to the methyl tertiary butyl ether is 1:4-1:15; the volume ratio of the emulsion raw material, the dairy product or the blood plasma to the ultrapure water is 1:1-1:5.
The organic solvent A, the internal standard solution B, the organic solvent C and the liquid D are selected and adjusted, and the selection and adjustment of the proportion ranges of the organic solvent A, the internal standard solution B, the organic solvent C and the liquid D are adopted, so that the extraction effect of the total lipid containing polar lipids is optimal under the condition of each proportion;
Optionally, the mass-to-volume ratio of the total lipid extract containing polar lipids to the organic solvent E is 30mg:1 mL-100 mg:1mL; the mass volume ratio of the total lipid extract containing polar lipids to the organic solvent F is 5mg:1 mL-25 mg:1mL; the mass volume ratio of the total lipid extract containing polar lipid to the organic solvent G is 10mg:1 mL-50 mg:1mL; the mass volume ratio of the total lipid extract containing polar lipid to the organic solvent H is 30mg:1 mL-90 mg:1mL; the mass volume ratio of the total lipid extract containing polar lipids to the organic solvent I is 5mg:1 mL-25 mg:1mL; the mass volume ratio of the total lipid extract containing polar lipids to the organic solvent J is 5 mg/1 mL-50 mg/1 mL.
Through the selection and adjustment of the organic solvent E, the organic solvent F, the solution G, the organic solvent H and the organic solvent I and the selection and adjustment of the proportion ranges, the enrichment of polar lipids containing polar lipids and the sample consumption for purification are less under the proportion conditions, and the enrichment of 14 large types of polar lipids can be realized.
The cholesterol, the ceramide and the phospholipid are quantitatively detected by adopting an internal standard method and combining a standard curve, a standard curve equation of standard concentration/internal standard substance concentration-standard concentration area/internal standard substance area is calculated, and a linear range and a correlation coefficient are calculated; the ganglioside is quantitatively detected by adopting an external standard method, and a linear equation, a linear range and a correlation coefficient are calculated.
Optionally, the ultrasonic conditions are 40kHz; the centrifugation conditions were 25℃and 8000rpm, and the centrifugation was carried out for 10min.
Optionally, the flow rate of the solid phase extraction elution is 0.03-0.1 mL/min, preferably 0.05mL/min.
Optionally, the polar lipid comprises: cholesterol, ceramide Cer, phosphatidylglycerol PG, phosphatidylethanolamine PE, phosphatidylinositol PI, lysophosphatidylethanolamine LPE, phosphatidic acid PA, monosialoganglioside GM 3, phosphatidylserine PS, phosphatidylcholine PC, bissialoganglioside GD 3, sphingomyelin SM, lysophosphatidylcholine LPC, and cardiolipin CL.
The invention has the beneficial effects that:
The detection method of the invention comprises the following steps: the method comprises the steps of extracting total lipid containing polar lipid, enriching and purifying the polar lipid to obtain polar lipid enriched material, and carrying out supercritical fluid chromatography-mass spectrometry detection analysis on the polar lipid enriched material after redissolving, so that enrichment of a sample with extremely low content (less than 5%) of the polar lipid is realized, the method is simple and convenient to operate, the sample consumption is low, the cost is low, the detection is rapid, the qualitative and quantitative capabilities are high, the practicability is high, the result is stable and reliable, and the method is suitable for uniformly detecting the polar lipid with wide polar range distribution in milk. The method can detect 14 major types of polar lipids within 10 minutes, and is suitable for rapid analysis of polar lipids in various emulsions, dairy products, milk powder and other biological samples. The result of the measurement by the internal standard method is accurate, and the relative values of the peak areas of the internal standard substance and the measured component are measured to calculate, so that errors caused by changes of operating conditions, loading volumes and the like are eliminated to a certain extent.
Drawings
The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate the invention and together with the description serve to explain, without limitation, the invention. In the drawings:
FIG. 1 is a graph showing the results of thin layer chromatography of the polar lipid enrichment and purification eluents of example 1, under the following conditions: 20 x 20cm silica gel glass plate, after spotting, in developing agent n-hexane: diethyl ether: acetic acid (80:20:1, v/v/v) and the plates were removed when the solvent front was extended 1cm from the front of the plates, dried and developed with iodine, as follows;
FIG. 2 is a plot of the results of thin layer chromatography of the polar lipid enrichment and purification of the eluents of example 3;
FIG. 3 is a plot of the results of thin layer chromatography of the polar lipid enrichment and purification of the eluents of example 5;
FIG. 4 is a total ion flow chromatogram of the supercritical fluid chromatography of example 1;
FIG. 5 is a total ion flow chromatogram of the supercritical fluid chromatography of example 2;
FIG. 6 is a total ion flow chromatogram of the supercritical fluid chromatography of example 3;
FIG. 7 is a total ion flow chromatogram of the supercritical fluid chromatography of example 4;
FIG. 8 is a total ion flow chromatogram of the supercritical fluid chromatography of example 5;
FIG. 9 is a total ion flow chromatogram of the supercritical fluid chromatography of example 6.
Detailed description of the preferred embodiments
The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto: the following detailed description of the invention will provide those skilled in the art with a more readily understood description of how the invention may be practiced. It should be understood that while the invention has been described in connection with its preferred embodiments, these embodiments are merely illustrative and are not intended to limit the scope of the invention.
The polar lipids in the present invention comprise the following 14 broad classes: cholesterol, ceramide (Cer), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylinositol (PI), lysophosphatidylethanolamine (LPE), phosphatidic Acid (PA), monosialoganglioside (GM 3), phosphatidylserine (PS), phosphatidylcholine (PC), bissialoganglioside (GD 3), sphingomyelin (SM), lysophosphatidylcholine (LPC), and Cardiolipin (CL).
The emulsion raw material in the invention refers to white or yellowish opaque liquid secreted by mammals such as human, cattle, sheep, donkey or camel from mammary glands; the "dairy product" refers to various foods in the form of liquid, powder, gel, paste, solid, concentrate, suspension or instant form which are prepared by using cow milk, sheep milk, donkey milk or camel milk and processed products thereof as main raw materials and adding or not adding proper amount of vitamins, minerals and other auxiliary materials.
Materials and reagents used in the invention:
Phosphatidylglycerol PG (16:0/18:1), phosphatidylethanol PE (14:0/14:0), phosphatidylinositol PI (16:1/16:0), lysophosphatidylethanolamine LPE (18:1/0:0), phosphatidic acid PA (16:0/18:1), monosialoganglioside GM 3, sphingomyelin SM (d18:1/23:0), lysophosphatidylcholine LPC (18:1/0:0) and bissialoganglioside GD 3 standards were all purchased from Sweden Larodan FINE CHEMICALS AB.
Ceramide Cer (d18:1/16:0), cardiolipin CL (14:0/14:0/14:0/14:0) standard: company Avanti Polar Lipids, U.S.; phosphatidylserine PS (14:0/14:0) standard: sigma Co., USA; cholesterol standard: aladin Corp; isotopic internal standard ceramide Cer (d18:1-D7/15:0), cardiolipin CL (18:2/18:2/18:2/18:2) -D5 and SPLASH LIPIDOMIX mix internal standards: silica gel solid phase extraction column of Avanti Polar Lipids company in U.S.: shanghai An Spectrum experiment science and technology company; chromatographic grade n-hexane: chinese Taitan technologies Co; chromatographic grade methanol: company TEDIA HIGH Purity Solvents, U.S.; chromatographic grade chloroform: chinese medicine reagent company; high purity carbon dioxide (purity not less than 99.999%): tin free Xin tin instruments gas company.
Supercritical fluid chromatography conditions in the examples: the column was an acquisition UPC 2 BEH (150 mm. Times.3.0 mm,1.7 μm, waters, USA); the column temperature is 55 ℃, the back pressure is 1500psi, and the sample injection volume is 2 mu L; eluent a is supercritical CO 2 (purity is more than or equal to 99.99%), eluent b is methanol/water solution (methanol volume percentage is 97%), 20mM ammonium formate is contained in eluent b, the flow rate of mobile phase is 1.1mL/min, the elution gradient is 0min,5% eluent b;5.5min,48% eluent b;10min,48% eluent b.
Mass spectrometry conditions in the examples: the flow rate of collision gas (argon) is 50L/h, the flow rate of desolvation gas (nitrogen) is 700L/h, the ion source temperature is 100 ℃, the desolvation temperature is 400 ℃, the taper hole voltage is 30eV, the low-energy collision is set to be 6eV, the high-energy collision is set to be 20-45 eV, and the mass-to-charge ratio scanning range is 100-1600 m/z.
The isotope internal standard mixed solution related in the invention comprises the following components:
cer (d18:1-D7/15:0): the sphingosine part in the molecular structure of the ceramide is C18:1 and contains 7 deuterium, and the fatty acid part is C15:0;
CL (18:2/18:2/18:2/18:2) -D5: all fatty acids in the cardiolipin molecular structure are C18:2, and the glycerol skeleton connecting two phosphate groups contains 5 deuterium;
cholesterol-D7: the cholesterol structure contains 7 deuterium;
PG (15:0/18:1-D7): two fatty acid side chains in the phosphatidylglycerol molecular structure are C15:0 and C18:1, wherein C18:1 contains 7 deuterium;
PE (15:0/18:1-D7): two fatty acid side chains in the phosphatidylethanolamine molecular structure are C15:0 and C18:1, wherein C18:1 contains 7 deuterium;
PI (15:0/18:1-D7): two fatty acid side chains in the phosphatidylinositol molecular structure are C15:0 and C18:1, wherein C18:1 contains 7 deuterium;
LPE 18:1-D7, wherein the fatty acid side chain in the molecular structure of the lysophosphatidylethanolamine is C18:1, and C18:1 contains 7 deuterium;
PA (15:0/18:1-D7): two fatty acid side chains in the phosphatidic acid molecular structure are C15:0 and C18:1, wherein C18:1 contains 7 deuterium;
PS (15:0/18:1-D7): two fatty acid side chains in the phosphatidylserine molecular structure are C15:0 and C18:1, wherein C18:1 contains 7 deuterium;
PC (15:0/18:1-D7): two fatty acid side chains in the phosphatidylcholine molecular structure are C15:0 and C18:1, wherein C18:1 contains 7 deuterium;
SM (d18:1-D9): the sphingosine part in the molecular structure of the ceramide is C18:1 and contains 9 deuterium;
LPC 18:1-D7: the fatty acid side chain in the molecular structure of the lysophosphatidylcholine is C18:1, wherein C18:1 contains 7 deuterium;
The invention is further illustrated by the following examples.
Example 1: detection of polar lipids in breast milk samples
1) Extraction of total lipid containing polar lipids:
Adding 10mL of methanol and 40 mu L of internal standard solution into 4mL of breast milk, shaking for 30 seconds, mixing uniformly, adding 20mL of methyl tertiary butyl ether, shaking for 15 minutes, mixing uniformly, performing ultrasonic ice-water bath for 10 minutes, adding 4.5mL of ultrapure water, promoting layering, shaking for 60 seconds, and mixing uniformly to obtain a mixture I.
Centrifuging the obtained mixture I by a centrifuge, removing the upper organic solution, and removing water and solvent by a vacuum centrifugal concentrator to obtain a total lipid extract containing polar lipids;
2) Enrichment and purification of polar lipids to obtain polar lipid concentrates:
firstly, carrying out activation treatment on a solid phase extraction column, wherein the filler of the solid phase extraction column is 500mg/3mL of silica gel, and the activation is realized by using n-hexane; 10mg of the total lipid extract containing polar lipids was dissolved in 100. Mu.L of chloroform: mixing methanol in the volume ratio of 8 to 1 to obtain sample liquid, and loading the sample liquid onto an activated solid phase extraction column;
1mL of n-hexane was used sequentially: the diethyl ether is mixed with 0.6mL of n-hexane according to the volume ratio of 50:1: eluting the column by diethyl ether according to a mixed solution with the volume ratio of 6:1 to obtain neutral fat eluent containing most of triglyceride;
This was followed by 0.5mL of n-hexane: the diethyl ether is mixed with 3mL of methanol and 1.5mL of chloroform according to the volume ratio of 1:1: methanol: eluting the column by water according to a mixed solution with the volume ratio of 3:5:2 to obtain an eluent rich in polar lipid, and drying by nitrogen to obtain a polar lipid enriched substance.
3) Performing supercritical fluid chromatography-mass spectrometry detection analysis on the polar lipid concentrate:
Cholesterol, ceramide (Cer), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylinositol (PI), lysophosphatidylethanolamine (LPE), phosphatidic Acid (PA), phosphatidylserine (PS), phosphatidylcholine (PC), sphingomyelin (SM) and Lysophosphatidylcholine (LPC) standard substances were prepared into a polar lipid mixed standard solution stock solution using chloroform methanol (2:1, v/v). The quantitative analysis is realized by combining an internal standard method with a concentration-peak area standard curve, and the quantitative analysis is performed by using Lipidblast and Progenesis MetScope self-built local databases in Progenesis QI software.
The results were as follows:
1. Recovery of solid phase extraction the recovery of polar lipid standards was determined by comparing the average peak areas of the polar lipids before and after solid phase extraction (6 replicates) and the results are shown in table 1. Thin layer chromatography results, see fig. 1, show that most of the triglycerides are effectively removed and cholesterol is retained in the components of the polar lipid concentrate.
TABLE 1 determination of polar lipid recovery
2. Standard curve equations for standard concentration/internal standard concentration-standard concentration area/internal standard area are calculated, and linear ranges and correlation coefficients are calculated. 3 parts of 10mg of breast milk polar lipid concentrate was taken as blank sample, and chloroform: the methanol is redissolved to 5mg/mL according to the volume ratio of 2:1, mixed standard solution with the content of 1.2 times, 1 time and 0.8 times of the content of each polar lipid in the polar lipid enrichment of breast milk and 40uL internal standard solution with the same amount are respectively added, the methodological verification is carried out, the parallelism is carried out for 3 times under each standard adding level, and the daily precision, the daily precision and the recovery rate of the method are calculated by combining a standard curve equation, and are shown in tables 2 and 3.
TABLE 2 standard curve equation for cholesterol, ceramide and phospholipid standards
TABLE 3 precision and labeling recovery of cholesterol, ceramide and phospholipid standards
3. The result of supercritical fluid chromatography is shown in figure 4. Mass spectrometry detection results: 187 kinds of polar lipids were co-detected in breast milk, and 8 kinds of Cardiolipin (CL) were detected by including 11.263mg/100mL of cholesterol, 7.813mg/100mL of ceramide (Cer), 0.018mg/100mL of Phosphatidylglycerol (PG), 2.154mg/100mL of Phosphatidylethanolamine (PE), 0.341mg/100mL of Phosphatidylinositol (PI), 0.546mg/100mL of Lysophosphatidylethanolamine (LPE), 0.118mg/100mL of Phosphatidic Acid (PA), 0.949mg/100mL of Phosphatidylserine (PS), 2.146mg/100mL of Phosphatidylcholine (PC), 8.541mg/100mL of Sphingomyelin (SM), 0.039mg/100mL of Lysophosphatidylcholine (LPC).
Example 2: detection of gangliosides in breast milk samples
1) Ganglioside enrichment extraction:
4mL of breast milk was added to example 1, followed by shaking for 30 seconds, and mixing was performed, followed by addition of 21.6mL of methanol and 10.8mL of chloroform. After gentle shaking at 150rpm for 30 minutes at 25 ℃, the resulting mixture was centrifuged at 8000rpm for 10 minutes at 25 ℃ with a centrifuge. The polar lipid-rich supernatant was collected and 4mL deionized water and 2mL chloroform were added to the lower precipitate: and (3) carrying out secondary extraction on the methanol according to the mixed solution with the volume ratio of 1:2. After gentle shaking at 150rpm for 30 minutes at 25 ℃, the mixture was centrifuged at 8000rpm for 10 minutes at 25 ℃ with a centrifuge. The polar lipid-rich supernatant was collected, and the two extracted polar lipid-rich supernatants were combined and added to 10.4mL of deionized water, followed by gentle inversion 4 times and centrifugation at 8000rpm for 10 minutes at 25℃in a centrifuge. Collecting supernatant rich in gangliosides. To the remaining mixture was added 4mL of potassium chloride solution at a concentration of 0.01mol/L and centrifuged at 8000rpm for 10 minutes at 25℃with a centrifuge, and a ganglioside-rich supernatant was collected. Combining the two supernatants rich in ganglioside, and blow drying with nitrogen to obtain ganglioside concentrate.
2) Performing supercritical fluid chromatography-mass spectrometry detection analysis on the obtained ganglioside enrichment:
Monosialoganglioside (GM 3) and bissialoganglioside (GD 3) standards were run with chloroform: and preparing a ganglioside mixed standard solution stock solution from the mixed solution of methanol according to the volume ratio of 2:1. Quantitative analysis is realized by adopting an external standard method, and Lipidblast and Progenesis MetScope in Progenesis QI software are used for self-building a local database for quantitative analysis.
The results were as follows:
1. 3 parts of ganglioside concentrate enriched from 4mL of breast milk were taken as blank samples, and 0.5mL of chloroform: and (3) re-dissolving the methanol according to a volume ratio of 2:1, respectively adding mixed standard solutions with high, medium and low concentration levels, wherein the content of the mixed standard solutions is about 1.2 times, 1 time and 0.8 time of the content of each ganglioside in the breast milk ganglioside enrichment, carrying out methodological verification, carrying out 3 times of parallelization under each standard adding level, and calculating the daily precision, the daily precision and the recovery rate of the method, wherein the table 3 and the table 4 are referred.
TABLE 3 Linear regression equation for gangliosides
Table 4 precision and labelling recovery of ganglioside standards
2. The result of supercritical fluid chromatography is shown in figure 5. Mass spectrometry detection results: monosialoganglioside (GM 3) 0.186mg/100mL, bissialoganglioside (GD 3) 0.666mg/100mL were detected in breast milk. The concentration of monosialoganglioside (GM 3) in breast milk was 3.5 times that of monosialoganglioside (GD 3).
Example 3: detection of polar lipids in camel milk samples
1) Extraction of total lipid containing polar lipids:
Adding 10mL of methanol and 40 mu L of internal standard solution into 4mL of camel milk, shaking for 30 seconds, mixing uniformly, adding 20mL of methyl tertiary butyl ether, shaking for 15 minutes to mix uniformly, performing ultrasonic ice-water bath for 10 minutes, adding 4.5mL of ultrapure water, promoting layering, shaking for 60 seconds, and mixing uniformly to obtain a mixture I.
Centrifuging the obtained mixture I by a centrifuge, removing the upper organic solution, and removing water and solvent by a vacuum centrifugal concentrator to obtain a total lipid extract containing polar lipids;
2) Enrichment and purification of polar lipids to obtain polar lipid concentrate:
Firstly, carrying out activation treatment on a solid phase extraction column, wherein the filler of the solid phase extraction column is 1g/6mL of silica gel, and the activation is realized by using n-hexane; 55mg of the total lipid extract containing polar lipids was dissolved in 1mL of chloroform: the methanol is mixed according to the volume ratio of 4:1 to obtain a sample loading liquid, and the sample loading liquid is loaded on an activated solid phase extraction column;
With 5mL of n-hexane: the diethyl ether is mixed with 3mL of normal hexane according to the volume ratio of 50:1: eluting the column by diethyl ether according to a mixed solution with the volume ratio of 4:1 to obtain neutral fat eluent containing most of triglyceride;
With 2mL of n-hexane: the diethyl ether is mixed with the volume ratio of 1:1, 4mL of methanol and 3mL of chloroform: methanol: eluting the column by water according to a mixed solution with the volume ratio of 3:5:2 to obtain an eluent rich in polar lipid, and drying by nitrogen to obtain a polar lipid enriched substance.
3) Performing supercritical fluid chromatography-mass spectrometry detection analysis on the polar lipid concentrate:
Cholesterol, ceramide (Cer), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylinositol (PI), lysophosphatidylethanolamine (LPE), phosphatidic Acid (PA), phosphatidylserine (PS), phosphatidylcholine (PC), sphingomyelin (SM) and Lysophosphatidylcholine (LPC) standard substances were prepared into a polar lipid mixed standard solution stock solution using chloroform methanol (2:1, v/v). The quantitative analysis is realized by combining an internal standard method with a concentration-peak area standard curve, and the quantitative analysis is performed by using Lipidblast and Progenesis MetScope self-built local databases in Progenesis QI software.
The results were as follows:
1. Thin layer chromatography results, see fig. 2, show that most of the triglycerides are effectively removed, but at the same time cholesterol is lost due to partial elution with neutral lipid.
2. As in example 1.
3. The result of supercritical fluid chromatography is shown in FIG. 6. Mass spectrometry detection results: in camel milk, 170 kinds of polar lipids were detected in total, and cholesterol contained 56.166mg/100mL, ceramide (Cer) 60.885mg/100mL, phosphatidylglycerol (PG) 0.200mg/100mL, phosphatidylethanolamine (PE) 108.151mg/100mL, phosphatidylinositol (PI) 1.603mg/100mL, lysophosphatidylethanolamine (LPE) 0.599mg/100mL, phosphatidic Acid (PA) 0.336mg/100mL, phosphatidylserine (PS) 7.704mg/100mL, phosphatidylcholine (PC) 40.923mg/100mL, sphingomyelin (SM) 62.292mg/100mL, lysophosphatidylcholine (LPC) 0.238mg/100mL, and Cardiolipin (CL) 7 kinds were detected, example 4: detection of polar lipids in milk fat globule membrane batch samples
1) Lipid extraction:
1g of formula milk powder is dissolved in 4mL of deionized water, 10mL of methanol and 40 mu L of internal standard solution are added, shaking is carried out for 30 seconds and mixing is carried out uniformly, 20mL of methyl tertiary butyl ether is added, shaking is carried out for 15 minutes until mixing is carried out uniformly, ultrasonic ice water bath is carried out for 10 minutes, 4.5mL of ultrapure water is added, layering is promoted, shaking is carried out for 60 seconds and mixing is carried out uniformly, and thus a mixture I is obtained.
Centrifuging the obtained mixture I by a centrifuge, removing the upper organic solution, and removing water and solvent by a vacuum centrifugal concentrator to obtain the total lipid extract containing polar lipid, wherein the content of triglyceride in the milk fat globule membrane ingredients is low, so that the total lipid extract can be used as a polar lipid enrichment.
2) The obtained polar lipid concentrate is subjected to supercritical fluid chromatography-mass spectrometry detection analysis:
Cholesterol, ceramide (Cer), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylinositol (PI), lysophosphatidylethanolamine (LPE), phosphatidic Acid (PA), phosphatidylserine (PS), phosphatidylcholine (PC), sphingomyelin (SM) and Lysophosphatidylcholine (LPC) standard substances were prepared into a polar lipid mixed standard solution stock solution using chloroform methanol (2:1, v/v). The quantitative analysis is realized by combining an internal standard method with a concentration-peak area standard curve, and the quantitative analysis is performed by using Lipidblast and Progenesis MetScope self-built local databases in Progenesis QI software.
The results were as follows:
1. as in example 1.
2. As in example 1.
3. The result of supercritical fluid chromatography is shown in FIG. 7. Mass spectrometry detection results: 146 polar lipids were detected in the milk fat globule membrane formulation, and cholesterol was 125.715mg/g, ceramide (Cer) 34.164mg/g, phosphatidylglycerol (PG) 0.706mg/g, phosphatidylethanolamine (PE) 363.542mg/g, phosphatidylinositol (PI) 12.130mg/g, lysophosphatidylethanolamine (LPE) 2.072mg/g, phosphatidic Acid (PA) 2.054mg/g, phosphatidylserine (PS) 55.555mg/g, phosphatidylcholine (PC) 103.587mg/g, sphingomyelin (SM) 184.074mg/g, lysophosphatidylcholine (LPC) 0.189mg/g, and Cardiolipin (CL) 4 species were detected.
Example 5: detection of polar lipids in a formula sample
1) Lipid extraction:
1g of formula milk powder is dissolved in 4mL of deionized water, 10mL of methanol and 40 mu L of internal standard solution are added, shaking is carried out for 30 seconds and mixing is carried out uniformly, 20mL of methyl tertiary butyl ether is added, shaking is carried out for 15 minutes until mixing is carried out uniformly, ultrasonic ice water bath is carried out for 10 minutes, 4.5mL of ultrapure water is added, layering is promoted, shaking is carried out for 60 seconds and mixing is carried out uniformly, and thus a mixture I is obtained.
Centrifuging the obtained mixture I by a centrifuge, removing the upper organic solution, and removing water and solvent by a vacuum centrifugal concentrator to obtain a total lipid extract containing polar lipids;
2) Enrichment and purification:
Firstly, carrying out activation treatment on a solid phase extraction column, wherein the filler of the solid phase extraction column is 1g/6mL of silica gel, and the activation is realized by using n-hexane; 50mg of the total lipid extract containing polar lipids was dissolved in 1mL of chloroform: the methanol is mixed according to the volume ratio of 4:1 to obtain a sample loading liquid, and the sample loading liquid is loaded on an activated solid phase extraction column;
With 5mL of n-hexane: the diethyl ether is mixed with 3mL of normal hexane according to the volume ratio of 50:1: eluting the column by diethyl ether according to a mixed solution with the volume ratio of 8:1 to obtain neutral fat eluent containing most of triglyceride;
1mL of n-hexane was used sequentially: the diethyl ether is mixed with the volume ratio of 1:1, 4mL of methanol and 3mL of chloroform: methanol: eluting the column by water according to a mixed solution with the volume ratio of 3:5:2 to obtain an eluent rich in polar lipid, and drying by nitrogen to obtain a polar lipid enriched substance.
3) Performing supercritical fluid chromatography-mass spectrometry detection analysis on the polar lipid concentrate:
Cholesterol, ceramide (Cer), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylinositol (PI), lysophosphatidylethanolamine (LPE), phosphatidic Acid (PA), phosphatidylserine (PS), phosphatidylcholine (PC), sphingomyelin (SM) and Lysophosphatidylcholine (LPC) standard substances were prepared into a polar lipid mixed standard solution stock solution using chloroform methanol (2:1, v/v). The quantitative analysis is realized by combining an internal standard method with a concentration-peak area standard curve, and the quantitative analysis is performed by using Lipidblast and Progenesis MetScope self-built local databases in Progenesis QI software.
The results were as follows:
1. As a result of thin layer chromatography, see fig. 3, cholesterol is shown to be retained in the fraction of polar lipids, but triglycerides are retained more in the polar lipid eluate.
2. Same as in example 1
3. The result of supercritical fluid chromatography is shown in FIG. 8. Mass spectrometry detection results: 207 polar lipids were co-detected in the formula, and contained cholesterol 1.311mg/g, ceramide (Cer) 1.224mg/g, phosphatidylglycerol (PG) 0.714mg/g, phosphatidylethanolamine (PE) 49.722mg/g, phosphatidylinositol (PI) 1.063mg/g, lysophosphatidylethanolamine (LPE) 0.413mg/g, phosphatidic Acid (PA) 0.810mg/g, phosphatidylserine (PS) 0.986mg/g, phosphatidylcholine (PC) 64.789mg/g, sphingomyelin (SM) 24.428mg/g, lysophosphatidylcholine (LPC) 0.509mg/g, and Cardiolipin (CL) 7 species were detected.
According to the preferred embodiments, the detection method disclosed by the invention realizes the uniform detection of the 14-class polar lipids with wide polar range distribution in milk in 10 minutes in all the preferred embodiments, and is rapid in detection, strong in qualitative and quantitative capability, strong in practicability, stable and reliable in result and suitable for detection. Comparative example 2, example 3 shows a similar neutral lipid removal effect to example 2 in enrichment and purification, but with a small loss of cholesterol; comparative example 2, example 5 has similar cholesterol retention capacity for enrichment and purification as example 2, but with more triglyceride residues. But in general, the solid phase extraction method used for enrichment and purification in the invention can be suitable for enrichment of samples with extremely low content (less than 5%) of polar lipids, and has the advantages of simple operation, low sample consumption and low cost.
Example 6: detection of polar lipids in blood samples
1) Lipid extraction:
To 100. Mu.L of plasma, 250. Mu.L of methanol and 10. Mu.L of an internal standard solution were added, followed by shaking for 30 seconds and mixing, and then 1mL of methyl tert-butyl ether was added, followed by shaking for 15 minutes to mixing, and then an ultrasonic ice-water bath was performed for 10 minutes, and then 0.35mL of ultra-pure water was added, and delamination was promoted and shaking for 60 seconds and mixing was performed to obtain a mixture I.
Centrifuging the obtained mixture I by a centrifuge, removing the upper organic solution, and removing water and solvent by a vacuum centrifugal concentrator to obtain a total lipid extract containing polar lipids; the triglyceride content in the blood sample is low, so that the blood sample can be used as a polar lipid enrichment.
4) The obtained polar lipid concentrate is subjected to supercritical fluid chromatography-mass spectrometry detection analysis:
Cholesterol, ceramide (Cer), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylinositol (PI), lysophosphatidylethanolamine (LPE), phosphatidic Acid (PA), phosphatidylserine (PS), phosphatidylcholine (PC), sphingomyelin (SM) and Lysophosphatidylcholine (LPC) standard substances were prepared into a polar lipid mixed standard solution stock solution using chloroform methanol (2:1, v/v). The quantitative analysis is realized by combining an internal standard method with a concentration-peak area standard curve, and the quantitative analysis is performed by using Lipidblast and Progenesis MetScope self-built local databases in Progenesis QI software.
The results were as follows:
1. as in example 1.
2. As in example 1.
3. The result of supercritical fluid chromatography is shown in FIG. 9. Mass spectrometry detection results: in plasma, 247 polar lipids were detected, which contained cholesterol 312.913. Mu.g/mL, ceramide (Cer) 20.686. Mu.g/mL, phosphatidylglycerol (PG) 0.235. Mu.g/mL, phosphatidylethanolamine (PE) 20.868. Mu.g/mL, phosphatidylinositol (PI) 33.487. Mu.g/mL, lysophosphatidylethanolamine (LPE) 3.909. Mu.g/mL, phosphatidic Acid (PA) 1.858. Mu.g/mL, phosphatidylserine (PS) 20.666. Mu.g/mL, phosphatidylcholine (PC) 814.709. Mu.g/mL, sphingomyelin (SM) 129.198. Mu.g/mL, cardiolipin (CL) 3.378. Mu.g/mL, lysophosphatidylcholine (LPC) 49.996. Mu.g/mL.
According to the preferred embodiments, the detection method disclosed by the invention realizes the uniform detection of the 14-class polar lipids with wide polar range distribution in milk in 10 minutes in all the preferred embodiments, and is rapid in detection, strong in qualitative and quantitative capability, strong in practicability, stable and reliable in result and suitable for detection. Comparative example 2, example 3 shows a similar neutral lipid removal effect to example 2 in enrichment and purification, but with a small loss of cholesterol; comparative example 2, example 5 has similar cholesterol retention capacity for enrichment and purification as example 2, but with more triglyceride residues. But in general, the solid phase extraction method used for enrichment and purification in the invention can be suitable for enrichment of samples with extremely low content (less than 5%) of polar lipids, and has the advantages of simple operation, low sample consumption and low cost.
The foregoing description of the preferred embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify for specific embodiments and applications without departing from the true spirit and scope of the present invention, and therefore, all such modifications, equivalents, and improvements that fall within the true spirit and scope of the present invention should be considered to be within the scope of the following claims.

Claims (6)

1. A method for quantitatively determining polar lipids, comprising the steps of:
(1) Extracting total lipid containing polar lipids:
Adding an organic solvent A into emulsion raw materials, dairy products or plasma, shaking and mixing uniformly, adding an internal standard solution B, adding an organic solvent C, shaking and mixing uniformly, then carrying out ultrasonic treatment, carrying out ice water bath ultrasonic treatment for 5-20 minutes, adding a liquid D, shaking and mixing uniformly, and promoting layering to obtain a mixture I;
Centrifuging the obtained mixture I, removing the upper organic solution, and removing water and solvent by using a vacuum centrifugal concentrator to obtain a total lipid extract containing polar lipids;
Wherein the organic solvent A is methanol; the internal standard solution B is an isotope internal standard mixed solution, and the organic solvent C is methyl tertiary butyl ether; the liquid D is ultrapure water;
(2) Enriching and purifying polar lipids:
Firstly, carrying out activation treatment on a solid phase extraction column, wherein the mass of silica gel filled in the solid phase extraction column is 200-1000 mg, the volume of the column is 3-6 mL, and n-hexane is used for activation;
Dissolving 5-200 mg of the total lipid extract containing polar lipids in an organic solvent E to obtain a sample solution, and loading the sample solution onto an activated solid phase extraction column;
Eluting the column with an organic solvent F and an organic solvent G in sequence to obtain neutral fat eluent containing triglyceride;
eluting the column with an organic solvent H, an organic solvent I and an organic solvent J in sequence to obtain an eluent rich in polar lipids, and drying with nitrogen to obtain a polar lipid enriched substance;
Wherein, the organic solvent E is chloroform: methanol is mixed according to the volume ratio of 1:1-8:1; the organic solvent F is n-hexane: the diethyl ether is mixed solution with the volume ratio of 100:1-5:1; the organic solvent G is n-hexane: the diethyl ether is mixed solution with the volume ratio of 10:1-2:1; the organic solvent H is n-hexane: the diethyl ether is mixed solution with the volume ratio of 3:1-0.5:1; the organic solvent I is methanol; the organic solvent J is chloroform: methanol: mixing liquid with water according to the volume ratio of 3:5:2;
(3) After redissolving the polar lipid enriched material, carrying out supercritical fluid chromatography-mass spectrometry detection analysis:
The redissolution solvent used for redissolution of the polar lipid enrichment is chloroform: the mass volume ratio of the polar lipid enrichment to the redissolution solvent is 1.5mg to 1mL to 10mg to 1mL according to the volume ratio of the mixed solution of methanol 2:1;
The conditions for the supercritical fluid chromatography-mass spectrometry detection analysis are as follows:
Supercritical fluid chromatography with water-based ultra-high performance liquid chromatography UPC 2, water-based UPC 2 BEH,150mm×3.0mm,1.7 μm, water, USA; the column temperature is 55 ℃, the back pressure is 1500psi, and the sample injection volume is 2 mu L; the eluent A is supercritical CO 2, the purity is more than or equal to 99.99%, the eluent B is methanol/water solution containing ammonium formate, the volume percentage of the methanol is 90% -100%, the content of the ammonium formate is 5 mM-35 mM, the elution gradient is 0min, and the content of the ammonium formate is 5% B;5.5min,48% B;10min,48% b;
The mass spectrum adopts WATERS SYNAPT Q-TOF-MS mass spectrometer, the flow rate of collision gas is 50L/h, the flow rate of desolvation gas is 700L/h, the temperature of an ion source is 100 ℃, the desolvation temperature is 400 ℃, the taper hole voltage is 30eV, the low-energy collision is set to be 6eV, the high-energy collision is set to be 20-45eV, and the scanning range of mass-to-charge ratio is 100-1600m/z;
the collision gas is argon;
the desolvation gas is nitrogen;
The isotope internal standard mixed solution contains: 30 μg/mL Cer (d18:1-D7/15:0), 5 μg/mL CL (18:2/18:2/18:2/18:2) -D5, 100 μg/mL cholesterol -D7,30μg/mL PG(15:0/18:1-D7),5μg/mLPE(15:0/18:1-D7),10μg/mL PI(15:0/18:1-D7),5μg/mL LPE 18:1-D7,7μg/mL PA(15:0/18:1-D7),5μg/mL PS(15:0/18:1-D7),160μg/mL PC(15:0/18:1-D7),30μg/mL SM(d18:1-D9) and 25 μg/mL LPC 18:1-D7;
The mass volume ratio of the total lipid extract containing polar lipids to the organic solvent E is 30mg:1 mL-100 mg:1mL; the mass volume ratio of the total lipid extract containing polar lipids to the organic solvent F is 5mg:1 mL-25 mg:1mL; the mass volume ratio of the total lipid extract containing polar lipid to the organic solvent G is 10mg:1 mL-50 mg:1mL; the mass volume ratio of the total lipid extract containing polar lipid to the organic solvent H is 30mg:1 mL-90 mg:1mL; the mass volume ratio of the total lipid extract containing polar lipids to the organic solvent I is 5mg:1 mL-25 mg:1mL; the mass volume ratio of the total lipid extract containing polar lipids to the organic solvent J is 5 mg/1 mL-50 mg/1 mL.
2. The method for quantitatively determining a polar lipid according to claim 1, wherein the volume ratio of the emulsion raw material, the dairy product or the plasma to the methanol is 1:2 to 1:8; the volume ratio of the emulsion raw material, the dairy product or the blood plasma to the internal standard solution B is 20:1-80:1; the volume ratio of the emulsion raw material, the dairy product or the blood plasma to the methyl tertiary butyl ether is 1:4-1:15; the volume ratio of the emulsion raw material, the dairy product or the blood plasma to the ultrapure water is 1:1-1:5.
3. The method for quantitatively determining a polar lipid according to claim 1, wherein the polar lipid comprises: cholesterol, ceramide Cer, phosphatidylglycerol PG, phosphatidylethanolamine PE, phosphatidylinositol PI, lysophosphatidylethanolamine LPE, phosphatidic acid PA, monosialoganglioside GM 3, phosphatidylserine PS, phosphatidylcholine PC, bissialoganglioside GD 3, sphingomyelin SM, lysophosphatidylcholine LPC, and cardiolipin CL.
4. The method for quantitative determination of polar lipids according to claim 1, wherein said ultrasonic conditions are 40kHz; the centrifugation conditions were 25℃and 8000rpm, and the centrifugation was carried out for 10min.
5. The method for quantitatively determining a polar lipid according to claim 1, wherein the flow rate at the time of solid phase extraction elution is 0.03 to 0.1mL/min.
6. Use of a method for quantitative determination of polar lipids according to any of claims 1 to 5 in emulsion raw materials, dairy products or plasma samples.
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